The ubiquitin proteasome system (“UPS”) is essential for the turnover of almost all cellular proteins, maintaining homeostatic levels in normal cells while controlling levels of oncogenes and tumor suppressors in transformed cells. In an ATP-dependent process, ubiquitin is transferred from the ubiquitin-activating enzyme E1 to the ubiquitin-conjugating enzyme E2, and covalently attached via an isopeptide linkage to a target protein bound to a ubiquitin ligase E3 (Ciechanover, “Proteolysis: from the lysosome to ubiquitin and the proteasome,”. Nat. Rev. Mol. Cell. Biol. 6:79-87 (2005)). Chains of four or more ubiquitin domains trigger degradation by the 26S proteasome.
FDA approval of the proteasome inhibitor Bortezomib (Velcade™, Millenium Pharmaceuticals Inc.) established the UPS as a validated target for treatment of multiple myeloma and mantle cell lymphoma (Bross et al., “Approval Summary for Bortezomib for Injection in the Treatment of Multiple Myeloma,” Clin. Cancer Res. 10:3954-3964 (2004) and Kane at al., “Bortezomib for the Treatment of Mantle Cell Lymphoma,” Clin. Cancer Res. 13:5291-5294 (2007)). Yet, advances in the clinical use of Bortezomib for solid tumors are lacking, resistance is developing, and peripheral neuropathy is a major side effect (Argyriou et al., “Bortezomib-induced Peripheral Neuropathy in Multiple Myeloma: A Comprehensive Review of the Literature,” Blood 112:1593-1599 (2008) and Orlowski et. al., “Proteasome Inhibitors In Cancer Therapy: Lessons from the First Decade,” Clin. Cancer Res. 14:1649-1657 (2008)). Recent investigations are now focused on inhibiting UPS proteins upstream of the proteasome (Ceccarelli et al., “An Allosteric Inhibitor of the Human Cdc34 Ubiquitin-conjugating Enzyme,” Cell 145:1075-1087 (2012); Orlicky et al., “An Allosteric Inhibitor of Substrate Recognition by the SCF(Cdc4) Ubiquitin Ligase,” Nat. Biotechnol. 28:733-737 (2010); and Soucy et al., “An Inhibitor of NEDD8-activating Enzyme as a New Approach to Treat Cancer,” Nature 458:732-736 (2009)). Of particular interest are inhibitors specific to E3 ligases in the hope of reducing off-target effects.
The Skp1-Cullin1-F-box (“SCF”) family is a multi-protein RING-finger E3 ligase that drives each stage of the cell cycle by controlling the protein levels of cyclins and cyclin-dependent kinase inhibitors (“CKI”s) (Cardozo et al., “The SCF Ubiquitin Ligase: Insights into a Molecular Machine,” Nat. Rev. Mol. Cell Biol. 5:739-751 (2004)). Through a coordinated repertoire of protein-protein interactions, the scaffold protein Cullin-1 (“Cul1”) binds both the Ring-box protein 1 (“Rbx1”), recruiting the E2-ubiquitin complex, and the adaptor protein Skp1, recruiting the F-Box E3 ligase (Petroski et al., “Function and Regulation of Cullin-RING Ubiquitin Ligases,” Nat. Rev. Mol. Cell Biol. 6:9-20 (2005)). The F-box family members dictate the substrate by binding a degron that is usually, but not always, post-translationally modified (Skowyra et al., “F-box Proteins are Receptors that Recruit Phosphorylated Substrates to the SCF Ubiquitin-ligase Complex,” Cell 91:209-219 (1997)).
The F-box protein S-phase kinase-associated protein 2 (“Skp2”) is overexpressed in human cancers and implicated in multiple murine models (Frescas et al., “Deregulated Proteolysis by the F-box Proteins SKP2 and Beta-TrCP: Tipping the Scales of Cancer,” Nat. Rev. Cancer 8:438-449 (2008); Lin et al., “Skp2 Targeting Suppresses Tumorigenesis by Arf-p53-independent Cellular Senescence,” Nature 464:374-379 (2010); and Nakayama et al., “Ubiquitin Ligases: Cell-cycle Control and Cancer,” Nat. Rev. Cancer 6:369-381 (2006)). SCF-Skp2 degrades known tumor suppressors CKIs p27, p21, and p57 (Carrano et al., “SKP2 is Required for Ubiquitin-mediated Degradation of the CDK Inhibitor p27,” Nat. Cell. Biol. 1:193-199 (1999); Kamura et al., “Degradation of p57Kip2 Mediated by SCFSkp2-dependent Ubiquitylation,” Proc. Natl. Acad. Sci. USA 100:10231-10236 (2003); and Yu et al., “Human CUL-1 Associates with the SKP1/SKP2 Complex and Regulates p21(CIP1/WAF1) and Cyclin D Proteins,” Proc. Natl. Acad. Sci. USA 95:11324-11329 (1998)). Recognition of the p27 degron is unique, being bound by a complex consisting of Skp2 and an accessory protein, Cdc kinase subunit 1 (Cks1), after phosphorylation of Thr-187 by CyclinE-CDK2 (Ganoth et al., “The Cell-cycle Regulatory Protein Cks1 is Required for SCF(Skp2)-mediated Ubiquitinylation of p27,” Nat. Cell Biol. 3:321-324 (2001); Montagnoli et al., “Ubiquitination of p27 is Regulated by Cdk-dependent Phosphorylation and Trimeric Complex Formation,” Genes Dev. 13:1181-1189 (1999); and Tsvetkov et al. “p27(Kip1) Ubiquitination and Degradation is Regulated by the SCF(Skp2) Complex through Phosphorylated Thr187 in p27,” Curr. Biol. 9:661-664 (1999)). Additional non-phosphorylated residues of the p27 degron reinforce this trimeric complex for a high rate of p27 ubiquitylation (Hao et al., “Structural Basis of the Cks1-dependent Recognition of p27(Kip1) by the SCF(Skp2) Ubiquitin Ligase,” Mol. Cell 20:9-19 (2005); Sitry et al., “Three Different Binding Sites of Cks1 are Required for p27-ubiquitin Ligation,” J. Biol. Chem. 277:42233-42240 (2002); Wang et al., “A Negatively Charged Amino Acid in Skp2 is Required for Skp2-Cks1 Interaction and Ubiquitination of p27Kip1,” J. Biol. Chem. 278:32390-32396 (2003); and Wang et al., “Molecular and Biochemical Characterization of the Skp2-Cks1 Binding Interface,” J. Biol. Chem. 279:51362-51369 (2004)).
Small molecule inhibitors have been successfully developed against E3 ligase-substrate interfaces, including Mdm2-p53 and IAPs-caspases (Vassilev et al., “In Vivo Activation of the p53 Pathway by Small-molecule Antagonists of MDM2,” Science 303:844-848 (2004) and Wang et al., “Cellular, Biochemical, and Genetic Analysis of Mechanism of Small Molecule IAP Inhibitors,” J. Biol. Chem. 279:48168-48176 (2004)). In addition, high-throughput screens designed to detect small molecules that stabilize p27 identified compounds that either inhibited 26S proteasome activity, prevented Skp2 from incorporating into the SCF complex, or downregulated Skp2 mRNA (Chen et al., “Targeting the p27 E3 Ligase SCF(Skp2) Results in p27- and Skp2-mediated Cell-cycle Arrest and Activation of Autophagy,” Blood 111:4690-4699 (2008); Nickeleit et al., “Argyrin A Reveals a Critical Role for the Tumor Suppressor Protein p27(kip1) in Mediating Antitumor Activities in Response to Proteasome Inhibition,” Cancer Cell 14:23-35 (2008); and Rico-Bautista et al., “Chemical Genetics Approach to Restoring p27Kip1 Reveals Novel Compounds with Antiproliferative Activity in Prostate Cancer Cells,” BMC Biol. 8:153 (2010)). No inhibitors specifically and directly targeted to the E3 ligase activity of Skp2 have been identified, however.
The present invention is directed to overcoming these and other deficiencies in the art.